U.S. patent number 10,155,221 [Application Number 15/996,853] was granted by the patent office on 2018-12-18 for high-throughput crystallographic screening device and method for crystalizing membrane proteins using a sub physiological resting membrane potential across a lipid matrix of variable composition.
This patent grant is currently assigned to University of Puerto Rico. The grantee listed for this patent is Carlos Baez-Pagan, Jose A. Lasalde-Dominicci, Orestes Quesada-Gonzalez, Josue Rodriguez-Cordero. Invention is credited to Carlos Baez-Pagan, Jose A. Lasalde-Dominicci, Orestes Quesada-Gonzalez, Josue Rodriguez-Cordero.
United States Patent |
10,155,221 |
Lasalde-Dominicci , et
al. |
December 18, 2018 |
High-throughput crystallographic screening device and method for
crystalizing membrane proteins using a sub physiological resting
membrane potential across a lipid matrix of variable
composition
Abstract
The invention is a high-throughput voltage screening
crystallographic device and methodology that uses multiple micro
wells and electric circuits capable of assaying different
crystallization condition for the same or different proteins of
interest at the same of different voltages under a humidity and
temperature controlled environment. The protein is solubilized in a
lipid matrix similar to the lipid composition of the protein in the
native environment to ensure stability of the protein during
crystallization. The invention provides a system and method where
the protein is transferred to a lipid matrix that holds a resting
membrane potential, which reduces the degree of conformational
freedom of the protein. The invention overcomes the majority of the
difficulties associated with vapor diffusion techniques and
essentially reconstitutes the protein in its native lipid
environment under "cuasi" physiological conditions.
Inventors: |
Lasalde-Dominicci; Jose A. (San
Juan, PR), Quesada-Gonzalez; Orestes (Canovanas, PR),
Rodriguez-Cordero; Josue (Carolina, PR), Baez-Pagan;
Carlos (Carolina, PR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Lasalde-Dominicci; Jose A.
Quesada-Gonzalez; Orestes
Rodriguez-Cordero; Josue
Baez-Pagan; Carlos |
San Juan
Canovanas
Carolina
Carolina |
PR
PR
PR
PR |
US
US
US
US |
|
|
Assignee: |
University of Puerto Rico (San
Juan, PR)
|
Family
ID: |
64605473 |
Appl.
No.: |
15/996,853 |
Filed: |
June 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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15638276 |
Jun 29, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C30B
7/12 (20130101); B01L 3/50853 (20130101); G16C
10/00 (20190201); C12Y 304/24069 (20130101); C30B
29/58 (20130101); B01J 19/0046 (20130101); C07K
1/113 (20130101); C12N 9/54 (20130101); C30B
30/02 (20130101); C07K 1/306 (20130101); G16B
15/00 (20190201); B01L 3/06 (20130101); C07K
14/70571 (20130101); B01J 2219/00725 (20130101); B01J
2219/00756 (20130101); B01L 2300/046 (20130101); C07B
2200/13 (20130101); B01L 2400/0415 (20130101); B01J
2219/00317 (20130101); B01J 2219/00605 (20130101); C40B
40/10 (20130101); B01J 2219/00653 (20130101); B01J
2219/00853 (20130101) |
Current International
Class: |
C30B
7/12 (20060101); C07K 1/30 (20060101); B01L
3/06 (20060101); G06F 19/16 (20110101); C30B
29/58 (20060101); B01J 19/00 (20060101); G06F
19/00 (20180101); C40B 40/10 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Carina Pareja-Rivera, et al, Recent Advances in the Understanding
of the Influence of Electric and Magnetic Fields on Protein Crystal
Growth, Cryst. Growth Des., Dec. 6, 2016. cited by applicant .
Edith Flores-Hernandez, et al., An electrically assisted device for
protein crystallization in a vapor-diffusion setup, J. Appl. Cryst.
(2013). 46, 832-834. cited by applicant.
|
Primary Examiner: Kunemund; Robert M
Attorney, Agent or Firm: Hoglund & Pamias, PSC Rios;
Roberto J.
Government Interests
GOVERNMENT INTEREST
The claimed invention was made with U.S. Government support under
grant number R01 GM098343 awarded by the National Institutes of
Health (NIH). The government has certain rights in this invention.
Claims
We claim:
1. A method for crystalizing membrane proteins comprising: adding a
membrane protein sample to at least one holding well of a sample
holding layer having a plurality of holding wells, wherein each
holding well comprises a separate pair of electrodes and ensuring
the membrane protein sample is in contact with both electrodes;
adding a crystallization precipitant solution to at least one lid
well of a lid layer having a plurality of lid wells; positioning
said sample holding layer on top of said lid layer so that said at
least one holding well lies on top and directly facing said a least
one lid well a sample unit; and incubating said membrane protein
sample by applying a voltage to the pair of electrodes of said at
least one holding well at a predetermined temperature and at a
predetermined humidity until said membrane protein sample is
crystallized.
2. The method of claim 1, wherein said membrane protein sample
comprises a solubilized membrane protein complex.
3. The method of claim 2, wherein said solubilized membrane protein
complex is a solubilized protein-detergent complex.
4. The method of claim 1, wherein said membrane protein sample is
incubated by placing said sealed sample holding layer inside an
incubator that is maintained at said predetermined temperature
prior to placing the sealed sample holding layer inside said
incubator.
5. The method of claim 1, wherein said membrane protein sample is
purified prior to being added to said at least one holding
well.
6. The method of claim 1, wherein said membrane protein sample is
provided in a lipid matrix.
7. The method of claim 6, wherein said lipid matrix has a variable
impedance during the crystallization process.
8. The method of claim 1, wherein said membrane protein sample is
subjected to a physiological membrane voltage at the beginning of
the incubation process for an amount of time prior to being
subjected to a sub-physiological membrane voltage.
9. The method of claim 1, wherein said voltage is maintained
constant.
10. The method of claim 1, wherein said voltage has a variable
waveform.
11. The method of claim 1, wherein said voltage is selected from a
sub-physiological voltage, a physiological voltage and a
supra-physiological voltage.
12. The method of claim 1, wherein said membrane protein sample
comprises a fluorescent tagged membrane protein.
13. The method of claim 1, further comprising performing in situ
X-ray diffraction experiments on said membrane protein sample.
14. The method of claim 1, further comprising performing
Fluorescence Recovery After Photobleaching experiments on said
membrane protein sample.
15. The method of claim 6, wherein said lipid matrix has a lipid
composition similar to the lipid composition of the membrane
protein in the native environment or is varied by lipid doping.
16. The method of claim 6, wherein pH and ionic content of the
lipid matrix is adjusted prior to crystallization.
17. The method of claim 1, wherein the same voltage is applied to
the pair of electrodes of all holding wells of said plurality of
holding wells.
18. The method of claim 1, wherein different voltages are applied
to the pair of electrodes of different holding wells of said
plurality of holding wells.
19. The method of claim 1, wherein said plurality of holding wells
contain membrane protein samples of the same member protein.
20. The method of claim 1, wherein said plurality of holding wells
contain membrane protein samples of different member proteins.
21. The method of claim 1, wherein the membrane protein samples on
said plurality of holding wells have the same protein
concentration.
22. The method of claim 1, wherein the membrane protein samples on
said plurality of holding wells have different protein
concentrations.
23. The method of claim 1, further comprising incubating a
plurality of samples units simultaneously.
24. The method of claim 1, wherein said lid layer comprises a
plurality of lid wells having the same geometric configuration as
said holding wells.
25. The method of claim 1, wherein the plurality of holding wells
has the same geometric shape or different geometric shapes.
26. The method of claim 1, wherein the pair of electrodes have the
same geometric shape in every holding well.
27. The method of claim 1, wherein at least one holding well has a
pair of electrodes having a geometric shape different than the pair
of electrodes of said plurality of holding wells.
28. The method of claim 1, wherein the holding wells have the same
volume or different volumes.
29. The method of claim 1, wherein said voltage is a D.C.
voltage.
30. The method of claim 11, wherein said sub-physiological voltage,
said physiological voltage and said supra-physiological voltage are
D.C. voltages.
Description
BACKGROUND OF THE INVENTION
Approximately 30% of the human genes code for membrane proteins.
Despite the efforts made by the best worldwide crystallographers,
only minute fraction of the entries in the Protein Data Bank
correspond to membrane proteins. A 3D protein structure is critical
to the advancement and efficiency of rational drug design, as well
as to protein structure-function studies, because the majority of
drugs and natural effector molecules stereo-specifically interact
with target proteins to affect their physiological and biological
activity by blocking or altering its properties.
Membrane proteins have hydrophobic domains and are expressed at
relatively low levels. This creates difficulties in obtaining
enough protein and growing crystals. The determination of
high-resolution structures for these proteins is far more difficult
than globular proteins. Nowadays, less than 0.1% of protein
structures determined are membrane proteins.
The crystallization process completely depends on the organization
ability of the proteins in a medium. Once these proteins are
organized repetitively in a solid three-dimensional lattice, it is
that the crystal of the protein is formed. This process is
regulated by physical-chemical, kinetic and thermodynamic factors
and consists of two steps. The first step is known as nucleation,
in which the protein molecules that are dissolved in the matrix
originally used to collect it from their natural environment, begin
to cluster. This gives rise to an extremely small focus, nucleus,
on the solution where there is a higher concentration of the
protein as a solute. The second step is the continuous and orderly
growth of this small focus of crystals. Nucleation can be initiated
by the inclusion of a precipitating agent as is the case in the
vapor diffusion technique.
During these processes the proteins could diffused and grouped
according to the conformation that it acquires both in the
extraction matrix used for its production and in the medium in
which it is being precipitated. Therefore, the crystals that form,
if this occurs, do not necessarily reflect the "true" structure of
these proteins in their natural environment. Specifically, many
technical problems are associated with the task of membrane protein
crystallization. The principal problem with the crystallization of
membrane proteins is that they are difficult to handle and
solubilize from its native environment in such a way that retains
native conformation and activity. Then, the solubilized
protein-detergent complex needs to be placed in an environment
similar to the native membrane and force nucleation. Membrane
proteins are inherently amphiphilic, they comprise hydrophobic and
hydrophilic regions. Due to their amphiphilic nature, membrane
proteins tend to aggregate rapidly to minimize the hydrophobic
regions. The addition of precipitants often causes an interaction
with the solubilized protein-detergent complex that induces phase
separation. For several decades the crystallization of membrane
proteins has been done using vapor diffusion methods including
hanging drop and sitting drop. The majority of the crystallization
methods using vapor diffusion techniques rely on reducing the
solubility of proteins in an aqueous environment, for instance
isoelectric focusing methods.
All membrane proteins are embedded inside a lipid membrane that
holds a resting membrane potential (RMP). On the basis of this
fundamental principle, we believe that the structural conformations
of membrane proteins (including ligand gated channels) are
voltage-dependent. The most remarkable example for the
voltage-dependent conformation of a protein is the large family of
voltage-dependent ion channels. Our group further studied this
concept while recoding single channel currents (cell-attached) in
myocytes. In order to estimate the opening and closing rate
constants (at -80 mV), it was necessary to record at least 100
bursts per acetylcholine concentration [ACh]. At high ACh
concentrations (>500 .mu.M) the number of bursts per [ACh] was
dramatically reduced as a result of desensitization. To overcome
this problem, we made a quick change in the polarity of the
amplifier (from -80 mV to +80 mV and back to -80 mV in .about.1
sec) and the single burst activity recovered immediately. This
experiment revealed that at +80 mV the agonist was expelled from
the ACh binding site and the channel conformation shifted from the
desensitized conformation and immediately equilibrated between the
open and closed states until it desensitized again. Thus, even in a
ligand-gated channel such as the nAChR, the desensitized
conformation can be reversed by changing the RPM. The biophysical
principle here is that a membrane protein sits in a voltage
gradient across a membrane and some localized domains in the
protein can display voltage dependency.
Accordingly, what is needed is a system and a method for the
crystallization of membrane proteins without the limitations and
constraints of the prior art systems and techniques including vapor
diffusion methods and LCP.
SUMMARY OF THE INVENTION
The invention is a high-throughput voltage screening
crystallographic device and methodology for protein crystallization
which consists of three layers of multiple micro wells electric
circuit capable of assaying different crystallization condition for
the same or different proteins of interest at different voltages
under a humidity and temperature controlled environment.
The methodology of the invention could be used with a single
micro-capillary crystal tube of different internal diameters
pre-cut for easy recovery of the protein crystal, or it can be
configured to allow multiple crystallization conditions in
parallel.
According to an aspect of the invention, the crystallization system
enables the use of close amphiphilic environments (e.g. monooelein)
for membrane protein crystallization and the rates of evaporation
are controlled by the relative humidity conditions, which are
adjusted in a precise and stable way during the combination of the
solubilized protein-detergent complex and amphiphilic reagents.
According to another aspect of the invention, the protein crystals
can nucleate and grow under different dehydration conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
Further features and advantages of the invention will become
apparent from the following detailed description taken in
conjunction with the accompanying figure showing illustrative
embodiments of the invention, in which:
FIG. 1 illustrates the components of the sample unit according to
an embodiment of the present invention.
FIG. 2 illustrates the sample unit partially assembled according to
an embodiment of the present invention.
FIG. 3 illustrates the sample unit assembled according to an
embodiment of the present invention.
FIG. 4 illustrates a side cross-sectional view of the sample unit
according to an embodiment of the present invention.
FIG. 5 illustrates a sample unit according to another embodiment of
the present invention.
FIG. 6 illustrates a crystallization system according to an
embodiment of the present invention.
FIG. 7 illustrates the sample unit preparation for a standard
protein sample loading according to an embodiment of the present
invention.
FIG. 8 illustrates the sample unit preparation for a vapor
diffusion protein sample loading according to an embodiment of the
present invention.
FIG. 9 shows images of protein samples without applying electric
potential.
FIG. 10 shows images of protein crystals for a fluorescence-labeled
protein according to an embodiment of the present invention.
FIG. 11 shows images of a plurality of nAChR crystals inside the
sample unit according to an embodiment of the present
invention.
FIG. 12 shows images of a plurality of nAChR crystals inside the
sample unit according to an embodiment of the present
invention.
FIG. 13 shows a confocal microscopy image of a nAChR-.alpha.BTX
crystal in the sample unit according to an embodiment of the
present invention.
FIG. 14 shows an image of a nAChR crystal in the loop according to
an embodiment of the present invention.
FIG. 15a shows an image of a nAChR crystal in the sample unit
according to an embodiment of the present invention.
FIG. 15b shows an image of a nAChR crystal in the loop according to
an embodiment of the present invention.
FIG. 16a shows an image of a nAChR crystal in the sample unit
according to an embodiment of the present invention.
FIG. 16b shows images of a nAChR crystal in the loop according to
an embodiment of the present invention.
FIG. 17 shows histograms for protein crystal formation frequency
distribution according to an embodiment of the present
invention.
FIG. 18 shows images of protein crystallization using
Voltage-Lipidic Cubic Phase-Fluorescence Recovery After
Photobleaching technique according to an embodiment of the present
invention.
FIG. 19 shows fractional fluorescence recovery and mobile fraction
plots for a Fluorescence Recovery After Photobleaching Assay
according to an embodiment of the present invention.
Throughout the figures, the same reference numbers and characters,
unless otherwise stated, are used to denote like elements,
components, portions or features of the illustrated embodiments.
The subject invention will be described in detail in conjunction
with the accompanying figures, in view of the illustrative
embodiments.
DETAILED DESCRIPTION OF THE INVENTION
The system of present invention provides a sample unit 1 including
a sample holding layer 1a and a lid layer 1b, wherein the sample
holding layer 1a includes at least one well 1c with a pair of
electrodes 1d and the lid layer 1b also includes at least one well
1e as shown in FIGS. 1-4. Alternatively, depending on the protein
loading method the lid layer can be substituted with thin strip of
clear adhesive tape. According to a preferred embodiment of the
invention, the volume per well in the sample holding layer is 50
.mu.l and 25 .mu.l in the lid layer well. However, other volumes
can also be used depending on several factors or parameters such as
but no limited to the amount of sample needed. As can be
appreciated, the wells have a rectangular shape with the dimensions
being selected based on the volume desired to hold the sample for
crystallization purposes. In accordance to another embodiment, the
wells are provided as round-shaped wells (FIG. 5) with the
dimensions also being selected based on the volume desired to hold
the sample for crystallization purposes. One advantage of using
round-shaped wells is that smaller-volume wells can be provided as
the electrodes can be positioned closer to each other requiring
smaller voltages and increasing the magnetic field effect during
crystallization.
FIG. 6 illustrates a general crystallization system according to
the present invention. An incubator 2 is provided to incubate the
sample unit 1 during the crystallization process. A power supply
unit 3 is connected to the electrodes of the sample holding layer
1a to provide the required voltage in accordance with the
invention. In addition, a temperature and humidity monitoring means
4 is provided to monitor and control the crystallization process
within specific temperature and humidity conditions. Other
monitoring and control means can be provided to ensure proper
crystallization of the proteins in accordance to the method of the
present invention. All these components can be provided externally
to the incubator 2 or alternatively can be integrated as part of
the incubator 2.
One important advantage of the invention is that multiple sample
units can be incubated simultaneously while applying a desired
voltage and maintaining specific incubation conditions. For
example, different protein concentrations can be provided in
different sample units or in different wells of the same sample
unit. Also, the system of the invention allows to supply the same
voltage to all the wells of the sample units or different voltages
can be supplied to each well. In addition, with proper control and
monitoring different crystallization techniques could be
simultaneously carried out in the incubator. The voltage supplied
to the sample units can be provided by a single voltage source or
via a multi-voltage source. This can be done using plural regulated
voltage sources or using a multi-voltage regulated output circuits.
It is important to note that the selection of regulated voltage
sources as well as the voltage ranges will depend on the type of
protein and membrane resistance which determines the range of
membrane potentials in which membrane protein crystals are formed
in a defined lipid matrix composition also using different
electrode diameters. In addition, the resistance of the lipid
membrane is critical to assure that ion flux is constant during the
crystallization process. Our data shows that crystallization of
membrane proteins can occur within a very limited range of sub
membrane potentials. As can be appreciated, the system of the
present invention is a higly-configurable and flexible system that
can be used with different protein crystallization methods and
overcomes the majority of the difficulties associated with the
typical methods.
In operation, the protein of interest needs to be extracted,
purified and properly prepared prior to loading into the sample
unit 1. Note that this step will vary depending on several factors
including but not limited to: the type and amount of protein,
physiological pH of the protein, ionic strength of the medium,
optimal crystallization potential, and solubility of the detergent
among others. For example, according to an embodiment of the
invention, Nicotinic Acetylcholine Receptor (nAChR) extraction was
performed by homogenizing 200 g of Torpedo californica tissue. To
perform the solubilization of the crude membranes, they were
thawed, and mixed with a 1% detergent solution containing
DB-1.times. Buffer (100 mM NaCl, 10 mM MOPS, 0.1 mM EDTA, and 0.02%
NaN3). The detergent used to extract the transmembrane proteins was
LysoFos Choline 16, Anagrade (LFC-16). After extraction,
purification step was carried out using affinity chromatography.
During the column's preparation, Bromoacetylcholine bromide was
coupled to Affigel 10 (Bio-Rad) with DB-1.times. as a coupling
buffer. The first step of the preparation of Affigel-10 was to
incorporate sulfhydryl groups. To do so, 25 ml of Affigel-10, to
which the conservator was eliminated through a series of washes in
isopropanol and water, was equilibrated with 50 ml of 20 mM MOPS at
pH 7.4. Afterwards 50 ml of cysteine 0.054M was added, allowing it
to react for one hour. After the cysteine excess was rinsed off
with 200 ml of water, 50 ml of the reducing agent dithiothreitol
(DTT) 0.1 M with MOPS at pH 8.0 was added for thirty minutes. After
equilibration using 100 ml of water, 500 mg of Bromoacetylcholine
bromide was added, which attached to the thiol groups in the gel.
The remaining thiol groups were blocked with 50 mg of
iodoacetamide. Once the Affigel-10 had undergone anhydrous
coupling, it was placed in an Econo Bio-Rad 1.5.times.20 cm column
and stored at 4.degree. C. with a low ionic force of 50 mM Sodium
Acetate pH 4.0. The solubilized extracts of crude membranes were
passed through the column, during which the nAChRs attached to the
acetylcholine by affinity. The elution of the chromatographic
matrix receptor was performed with a solution containing
carbamylcholine, which has greater affinity in the column. This
yields an elution solution containing purified nAChRs. All steps
were carried out in the cold room (4.degree. C.) or keeping the
samples on ice. As can be understood, one skill in the art would
know the exact conditions and parameters for protein extraction and
purification that would provide the optimal conditions for crystal
formation in accordance with the present invention.
Once the protein has been prepared, it needs to be loaded into the
sample units 1 prior to placement inside the incubator 2. This step
will vary depending on the type of crystallization desired. FIG. 7
illustrates the steps for a standard protein loading where the
crystallization precipitant solution is added to each well 1c of
the sample holding layer 1a and the protein sample is later
deposited in the center of each well 1c using a dispenser between
the circuit electrodes ensuring contact between the electrodes.
Afterwards, the sample unit is sealed with the lid layer 1b or
alternatively a layer of clear adhesive tape. For a vapor diffusion
protein loading, as shown in FIG. 8, the protein sample is
deposited in the center of each well 1c of the sample holding layer
1a between the circuit electrodes 1d, wherein the protein sample
must be in lipid cubic phase and must be in contact with both
circuit electrodes 1d. Then, the crystallization precipitant
solution is added to each well 1e of the lid layer 1b and finally
the the sample holding layer 1a is turned over or flipped
180.degree. and placed on top of the lid layer 1b to seal the
sample unit. It is important to point out that the incubator 2
should be at the desired temperature prior to loading the proteins
in the sample units.
The next step is to calibrate and prepare the system for incubation
during the crystallization process. To that effect, the voltage
source is turned on and adjusted to the maximum voltage value to be
applied to the sample units and then while measuring the voltage,
adjusting the output voltage to the desired values. Afterwards, the
voltage source is turned off and its output is electrically
connected to the electrodes arrangement on the sample units inside
the incubator 2. The voltage on each well Is measured and adjusted
accordingly to ensure the required voltage for crystallization,
wherein the incubator is finally closed with the sample units
inside ready for crystallization.
Finally, the proteins are incubated for a predetermined amount of
time, which according to a preferred embodiment of the invention is
between 1-2 weeks. When the crystallization process is finished the
voltage supply is turned off and disconnected from the sample units
holding the protein crystals for subsequent removal from the
incubator and crystal extraction for appropriate analysis.
There are several aspects of the system and methodology to consider
when using the present invention for protein crystallization.
First, the membrane protein sample to be crystallized in this
system must be highly pure to ensure optimal crystallization. Also,
the membrane protein is solubilized in a lipid matrix of variable
composition at a particular lipid to protein ratio and to ensure
stability of the membrane protein, the lipid composition used for
the crystallization must be similar to the lipid composition of the
protein in the native environment. To that effect, a lipidomic
analysis of the model membrane protein must be performed and a
lipid matrix containing lipid-detergent analogs similar to the
native lipid composition of the protein must be used. In addition,
a variety of lipid phases can be used with the invention, which in
turn results in a variable degree of hydration. Furthermore, the
lipid composition of the matrix can be variable depending on the
type of membrane protein sample. In an embodiment of the invention,
the resistance of the lipid matrix is in the range of
1-100M.OMEGA.. However, other ranges of resistances such as
25-200M.OMEGA. can be used depending on the protein size, protein
concentration and lipid composition. It is important to point out
that the resistance of the lipid matrix (LMx) remains variable in
the initial phases of the crystallization, however, it must reach a
constant value during the crystallization procedure (24-168 hours).
Optionally, at any given point during the crystallization
procedure, lipid doping can be performed depending on the
resistance of the lipid-protein matrix and the membrane protein.
Furthermore, a variable physiological membrane potential (-140
mv-10 mV) can be used to stabilize the membrane protein
conformation at the beginning of the experiment and after a period
of 1-2 hours the potential can be slowly decreased to reach a sub
physiological range of potential (-5 mV to -20 mV) where it can be
either kept constant or changed (voltage-ramp mode) for the
remaining period of the crystallization process. In addition, the
pH and ionic content of the lipid matrix can be manipulated during
crystallization. Membrane protein crystal formation occurs in a
time frame of 24-96 hours depending on the membrane protein
concentration and composition of the lipid matrix and the crystals
are produced at room temperature or, if necessary, at lower
temperatures. Also, lipid diffusion experiments can be performed to
optimize crystal formation and quality.
An important feature of the invention is that when using a
fluorescent tagged membrane protein the system will allow
monitoring crystal formation and membrane protein stability during
crystallization process. Moreover, Fluorescence Recovery After
Photobleaching (FRAP) experiments can be used during the
crystallization process to determine mobile fraction of the
membrane protein and to optimize the lipid composition of the lipid
matrix to achieve crystallization. It is important to point out
that mobile fraction of membrane proteins in the lipid matrix will
have to be over 75% to facilitate crystallization. Another
important feature of the invention is that the system allows
performing X-ray diffraction experiments in situ and that there is
no limitation in the molecular weight (MW) of the protein, thus
larger membrane complexes can be crystallized. This is extremely
important since the invention overcomes the MW weight limitation
that is intrinsic to the Lipidic Cubic Phase (LCP) methodology. The
membrane protein crystal is harvested while the protein is grown at
a sub-physiological membrane potential (-5 mV to -140 mV) and the
membrane protein crystal is immediately frozen at -80.degree.
C.
The effectiveness of the present invention will be now explained in
accordance to FIGS. 9-19.
FIG. 9 shows images of samples crystallization without applying
electric potential according to the present invention. As can be
appreciated, no well-formed crystals were found. In clear contrast
FIG. 10 shows the formation of protein crystals, where nAChR is
conjugated with a-Bungarotoxin Alexa Fluor.RTM. 488 for
fluorescence and was prepared using a lipid cubic phase (LCP)
technique (the arrows indicate the crystal formation). FIG. 11
shows several nAChR crystals growing in different sizes inside the
sample unit according the present invention. FIG. 12 shows nAChR
crystals formed from samples that were conjugated with .alpha.-BTX
and monoclonal antibodies. FIG. 13 shows a confocal microscopy
image of a well formed nAChR-.alpha.BTX crystal in the sample unit,
where the crystal structure can be appreciated. FIG. 14 shows an
image of a nAChR crystal (indicated by the arrow) in the loop ready
to be diffracted. FIGS. 15a and 15b show images of a nAChR crystal
in the sample unit (indicated by the arrow) and in the loop ready
to be diffracted, respectively. FIGS. 16a and 16b show additional
images of the nAChR crystal in the sample unit (indicated by the
arrow) and in the loop ready to be diffracted, respectively. FIG.
17 shows histograms (for raw and normalized data) of protein
crystal formation frequency distribution, where different voltages
where applied for the stimulation of crystal nucleation and the
sample size for the experiment was 160. FIG. 18 shows images of
Voltage-Lipidic Cubic Phase-
Fluorescence Recovery After Photobleaching Assay (V-LCP-FRAP) using
a lipid cubic phase (LCP) in which the sample is placed in a
lipidic and viscous environment. The Region of Interest (ROI) are
the areas in which fluorescence recovery is measured. This assay
was performed in order to determine which protein-detergent
complexes provide the highest protein stability, for structural
studies. FIG. 19 shows fractional fluorescence recovery and mobile
fraction graphs for a FRAP assay experiment where lipidic cubic
phase (LCP) was used. This experiment was carried out with the
implementation of monoclonal antibodies in monoolein matrix for the
nAChR-a-BTX complex using phospholipid analog detergent, where all
experiments were performed in triplicate and the incubation was
20.degree. C. and recorded every five days (three times for mAb-F8
and one time for mAb-B2).
A fundamental aspect of the present invention is the principle of
membrane resistance. To this effect, the conditions for membrane
protein crystal formation were assessed using a basic electrode
prototype to determine the range of membrane potentials in which
membrane protein crystals are formed in a defined lipid matrix
composition using different electrode diameters. In a lipid matrix
of define composition, crystal formation was observed within a
resistance range of 1-25 M.OMEGA. depending on the protein
concentration in the lipid matrix. The resistance range also
depends on the size and molecular weight of the proteins because
these are intrinsic parameters that affect the membrane
capacitance. The resistance of the lipid membrane is critical to
assure that ion flux is constant during the crystallization
process. Our data shows that crystallization of membrane proteins
can occur within a very limited range of sub membrane
potentials.
The present invention overcomes the majority of the difficulties
associated with vapor diffusion techniques (i.e, hanging drop,
sitting drop, etc.), because the protein-detergent complex is
rapidly mixed with a lipid matrix (LMx) of defined composition.
Second, the detergent is immediately diluted in an enriched lipid
matrix (LMx) where it diffuses from the protein-detergent complex
in a native hydrophobic/aqueous environment allowing critical
lipid-protein (and van Der Waals') interactions with the
hydrophobic domains of the membrane protein. Third, the dilution
and diffusion of the detergent from the protein-detergent complex
under the aforementioned conditions is critical to preserve
stability of the membrane protein and to reduce the aggregation
caused by denaturation of protein hydrophobic domains. Fourth,
during the process of detergent diffusion the membrane protein is
presumably preserved in a single conformation by the membrane
potential in LMx with constant resistance. Lastly, this methodology
essentially reconstitutes the membrane protein in its native lipid
environment under "cuasi" physiological conditions.
It is important to emphasize that the present invention provides a
system and method where the membrane protein is transferred to a
lipid matrix that holds a resting membrane potential, which reduces
the degree of conformational freedom of the protein. The system and
methodology led to consistent x-Ray diffractions from the
nAChR-LFC16 complex. The invention will serve to test new
approaches in a very challenging field of structural Biology and it
represents a step forward in the use of innovative approaches for
the solution membrane protein structures. This methodology was
developed after many attempts to crystallize the nAChR using vapor
diffusion methods and, more recently, LCP. Our team has being doing
electrophysiological recordings of nAChR channel activity for many
years and our basic understanding of the nAChR structure and
function was conceptualized in a physiological environment. The
system of the present invention was conceived to crystalize the
nAChR in its closest physiological environment, which includes a
native lipid composition and a fixed resting membrane
potential.
Although the invention has been described in conjunction with
specific embodiments, it is evident that many alternatives and
variations will be apparent to those skilled in the art in light of
the foregoing description. Accordingly, the invention is intended
to embrace all of the alternatives and variations that fall within
the spirit and scope of the invention.
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